RESOLUTION OF THE HETEROGENEOUS FLUORESCENCE IN MULTI-TRYPTOPHAN PROTEINS - ASCORBATE OXIDASE

Ascorbate oxidase is a copper-containing enzyme which catalyzes a redo x reaction between vitamin C and molecular oxygen. The protein, which shows a complex tertiary structure, is an homodimer of monomers, each containing three domains and 14 tryptophan residues. Recently, we have demonstrated by spectroscopic and ultracentrifugation techniques the existence of a stable dimeric intermediate along the unfolding pathway of this enzyme [Mei, G., Di Venere, A., Buganza, M., Vecchini, P., Ro sato, N. & Finazzi Agro, A. (1997) Biochemistry 36, 10917-10922]. In t his study, the steady-state and dynamic fluorescence features of ascor bate oxidase have been exploited in order to find a way of monitoring the individual subsystems of the protein. The fluorescence intensity a nd anisotropy upon excitation at 295 nm are extremely sensitive functi ons of the emission wavelength, indicating a great heterogeneity of th e system. The emission decay collected through a cut-off filter can be analyzed in terms of two continuous distributions of lifetimes. Using a monochromator in emission or an optical multichannel analyzer, the two distributions may be attributed to distinct components of the fluo rescence spectrum. Differential quenching by cesium chloride also conf irmed that the several tryptophan residues present in the protein stru cture may be grouped into two main classes, each with a different envi ronment. Once the complex fluorescence decay of ascorbate oxidase was analyzed and resolved, a comparison with the crystallographic data all owed a first, approximate attribution of the protein spectroscopic pro perties to some of the tryptophan residues. This might provide a power ful tool of investigation about the role of definite segments of the p rotein in its three-dimensional structure and catalytic activity. Furt hermore, the methodology set up for ascorbate oxidase can be usefully extended to other multitryptophan proteins.